Calcium ions (Ca2+) play a critical role in diverse physiological processes such as contraction, secretion, neurotransmission, gene transcription, and cell death. The voltage-gated calcium (Cav) channels open upon membrane depolarization, converting the membrane electrical signals to intracellular Ca2+-mediated events. Malfunction or dysregulation of Cav channels is associated with a broad spectrum of neurological, cardiovascular, and muscular disorders. Despite the physiological and pathophysiological significance of Cav channels, further progress has been acutely limited by the dearth of structural information. Indeed, the only available structure of any eukaryotic Cav channel is that of the Cav1.1 channel complex, which my group determined using single-particle electron cryo-microscopy (cryo-EM). Cav channels are targeted by multiple FDA-approved drugs for the treatment of neurological and cardiovascular disorders, and their activity is modulated by various peptide toxins. These ligands could be used to stabilize the Cav channels in various functional states, facilitating the dissection of the gating mechanism. In turn, structural elucidation of Cav channels in complex with the drugs and toxins will elucidate the molecular basis for their modes of action. These structures will guide mutagenesis for functional and mechanistic characterizations, serve as an important framework for homology modeling, ligand docking, and molecular dynamics simulation analyses, and eventually facilitate potential drug discovery. The overarching goal of this proposal is to achieve an improved mechanistic understanding of Cav channels through high-resolution structural determination of Cav1.1 in complex with various modulatory ligands using single-particle cryo-EM. In Aim 1, we will further improve the resolution of the Cav1.1 channel to beyond 3 by optimizing cryo-sample preparation and hardware configuration. Improved resolution will afford a more accurate structural template for molecular dynamics simulation analysis. In Aim 2, we will biochemically recapitulate the interactions between the purified Cav1.1 channel and various drugs and toxins, and elucidate the structures of Cav1.1 in complex with well-defined ligands. These structures will guide the design of mutations for functional characterizations and mechanistic investigations in cell-based electrophysiological assays. In Aim 3, we will investigate the structural basis for the modulation of Cav1.1 by the adaptor protein Stac3. This study will encompass crosslinking, mass spectrometric analysis, and new algorithms for cryo-EM to unravel the recognition between Stac3 and Cav1.1. Completion of the proposed research will advance our understanding of the function and disease-causing mechanisms of Cav channels as well as facilitate future drug discovery.